ABO Discrepancies & other problems Reneé Wilkins, PhD, MLS(ASCP)CM CLS 325/435 School of Health Related Professions University of Mississippi Medical Center ABO Discrepancies & other problems

Importance It is important to recognize discrepant results and how to (basically) resolve them Remember, the ABO system is the most important blood group system in relation to transfusions Misinterpreting ABO discrepancies could be life threatening to patients

Discrepancies A discrepancy occurs when the red cell testing does NOT match the serum testing results In other words, the forward does NOT match the reverse

Why? Reaction strengths could be weaker than expected Some reactions may be missing in the reverse or forward typings Extra reactions may occur

Patient Anti-A Anti-B A1 Cells B Cells 1 4+ 1+ 2 3 4 3+

What do you do? Identify the problem Most of the time, the problem is technical Mislabeled tube Failure to add reagent Either repeat test on same sample, request a new sample, or wash cells Other times, there is a real discrepancy due to problems with the patient’s red cells or serum

Discrepancy ? If a real discrepancy is encountered, the results must be recorded However, the interpretation is delayed until the discrepancy is RESOLVED

Errors

Technical Errors Clerical errors Reagent or equipment problems Mislabeled tubes Patient misidentification Inaccurate interpretations recorded Transcription error Computer entry error Reagent or equipment problems Using expired reagents Using an uncalibrated centrifuge Contaminated or hemolyzed reagents Incorrect storage temperatures Procedural errors Reagents not added Manufacturer’s directions not followed RBC suspensions incorrect concentration Cell buttons not resuspended before grading agglutination Clerical errors can result in fatalities. Please develop a set order when performing tests. This will eliminate mix-ups with tubes and prevent serious errors in reporting results.

Clotting deficiencies Serum that does not clot may be due to: Low platelet counts Anticoagulant therapy (Heparin, Aspirin, etc) Factor deficiencies Serum that does not clot completely before testing is prone to developing fibrin clots that may mimic agglutination Thrombin can be added to serum to activate clot formation Protamine sulfate can also be added to neutralize heparin in the serum.

Contaminated samples or reagents Sample contamination Microbial growth in tube Reagent contamination Bacterial growth causes cloudy or discolored appearance…do not use if you see this! Reagents contaminated with other reagents (don’t touch side of tube when dispensing) Saline should be changed regularly

Equipment problems Routine maintenance should be performed on a regular basis (daily, weekly, etc) Keep instruments like centrifuges, thermometers, and timers calibrated Uncalibrated serofuges can cause false results

Hemolysis Detected in serum after centrifugation (red) Important if not documented Can result from: Complement binding Anti-A, anti-B, anti-H, and anti-Lea Bacterial contamination Red supernatant

ABO discrepancies

ABO Discrepancies Problems with RBCs Problems with SERUM Weak-reacting/Missing antigens Extra antigens Mixed field reactions Problems with SERUM Weak-reacting/Missing antibodies Extra antibodies

Grouping Forward Reverse Missing/Weak Extra Mixed Field A/B Subgroup Disease (cancer) Acquired B B(A) Phenotype O Transfusion Bone Marrow Transplant Young Elderly Immunocompromised Cold Autoantibody Anti-A1 Rouleaux Alloantibody May cause all + reactions

Forward Grouping Problems

Red Cell Problems Affect the forward grouping results Missing or weak antigens Extra antigens Mixed field reactions

Forward Grouping: Missing or Weak antigens ABO Subgroups Disease (leukemia, Hodgkin’s disease) Anti-A Anti-B A1 Cells B Cells 4+ Group O Group A Since the forward and reverse don’t match, there must be a discrepancy (in this case, a missing antigen in the forward grouping)

Subgroups of A (or B) Subgroups of A account for a small portion of the A population (B subgroups rarer) These subgroups have less antigen sites on the surface of the red blood cell As a result, they show weakened (or missing) reactions when tested with commercial antisera Resolution: test with Anti-A1, Anti-H, and anti-A,B for A subgroups

Forward Grouping: Extra Antigens Acquired B B(A) phenotype Rouleaux Wharton’s Jelly Anti-A Anti-B A1 Cells B Cells 4+ 1+ EXAMPLE

Acquired B Phenotype Limited mainly to Group A1 individuals with: Lower GI tract disease Cancer of colon/rectum Intestinal obstruction Gram negative septicemia (i.e. E. coli)

Acquired B Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar…. Acquired B Phenotype Group A individual Galactosamine results from the deacetylating reaction, resembling D-galactose (found in Group B individuals). This sugar cross-reacts with the reagent anti-B, giving a weak reaction (but still technically it is “extra”). Patients should receive Group A units. Acquired B usually goes away when the condition resolves. N-acetyl galactosamine Galactosamine now resembles D-galactose (found in Group B) Bacterial enzyme removes acetyl group

Resolving Acquired B Check patient diagnosis: Infection? Some manufacturers produce anti-B reagent that does not react with acquired B Test patients serum with their own RBCs The patients own anti-B will not react with the acquired B antigen on their red cell (autologous testing)

B(A) phenotype Similar to acquired B Patient is Group B with an apparent extra A antigen The B gene transfers small amounts of the A sugar to the H antigen Sometimes certain anti-A reagents will detect these trace amount of A antigen Resolution: test with another anti-A reagent from another manufacturer

Other reasons for “extra” antigens Polyagglutination – agglutination of RBCs with human antisera no matter what blood type Due to bacterial infections Expression of hidden T antigens react with antisera Rouleaux – extra serum proteins Wharton’s Jelly – gelatinous substance derived from connective tissue that is found in cord blood and may cause false agglutination (Remember: only forward typing is performed on cord blood) Wash red cells or request new sample from heel, etc

Forward Grouping: Mixed Field Agglutination Results from two different cell populations Agglutinates are seen with a background of unagglutinated cells All groups transfused with Group O cells Bone marrow/stem cell recipients A3 phenotype (sometimes B3) Anti-A Anti-B A1 Cells B Cells 2+ mf 4+

Mixed Field Agglutination (Post transfusion) ~ (ABO Testing) Can be seen in A, B and AB individuals who have received O units. The antisera reacts with the patient’s RBCs, but not with the transfused O cells. ~ (Antibody screen) Can also be seen post transfusion if a person makes an antibody to antigen on donor cells; antibody agglutinates with donor cell, but not their on cells.

Reverse Grouping Problems

Reverse Grouping Affect the reverse grouping results Missing or weak antibodies Extra antibodies

Reverse Grouping: Missing or Weak antibodies Newborns Do not form antibodies until later Elderly Weakened antibody activity Hypogammaglobulinemia Little or no antibody production (i.e. immunocompromised) Often shows NO agglutination on reverse groupings

Resolving Weak or Missing antibodies Determine patients age, diagnosis Incubate serum testing for 15 minutes (RT) to enhance antibody reactions If negative, place serum testing at 4°C for 5 minutes with autologous control (Autocontrol, AC) This is called a “mini-cold” panel and should enhance the reactivity of the antibodies

Reverse Grouping: Extra Antibodies Cold antibodies (allo- or auto-) Cold antibodies may include anti-I, H, M, N, P, Lewis Rouleaux Anti-A1 in an A2 or A2B individual

Cold antibodies Sometimes a patient will develop cold-reacting allo- or auto-antibodies that appear as “extra” antibodies on reverse typing Alloantibodies are made against foreign red cells Autoantibodies are made against ones own red cells. Cold reacting antibodies cause agglutination with red cells at room temperature and below. The autocontrol will be positive. Resolution: warming tube to 37° and washing red cells can disperse agglutination; breaking the IgM bonds with 2-ME will also disperse cells

Rouleaux Can cause both extra antigens and extra antibodies “stack of coins” appearance May falsely appear as agglutination due to the increase of serum proteins (globulins) Stronger at IS and weak reaction at 37°C and no agglutination at AHG phase Associated with: Multiple meloma Waldenstrom’s macroglobulinemia (WM) Hydroxyethyl starch (HES), dextran, etc Agglutination at AHG should not occur because cells have been washed three times

Resolving Rouleaux Remove proteins! If the forward grouping is affected, wash cells to remove protein and repeat test If the reverse grouping is affected, perform saline replacement technique (more common) Cells (reagent) and serum (patient) centrifuged to allow antigen and antibody to react (if present) Serum is removed and replaced by an equal volume of saline (saline disperses cells)* Tube is mixed, centrifuged, and reexamined for agglutination (macro and micro)

Anti-A1 Sometimes A2 (or A2B) individuals will develop an anti-A1 antibody A2 (or A2B) individuals have less antigen sites than A1 individuals The antibody is a naturally occurring IgM Reacts with A1 Cells, but not A2 Cells + A1 cells AGGLUTINATION Anti-A1 from patient + A2 cells NO AGGLUTINATION

Resolving anti-A1 discrepancy 2 steps: Typing patient RBCs with Anti-A1 lectin Repeat reverse grouping with A2 Cells instead of A1 Cells Both results should yield NO agglutination Anti-A Anti-B A1 Cells B Cells 4+ 2+

Others… The Bombay phenotype (extremely RARE) results when hh is inherited These individuals do not have any antigens and naturally produce, anti-A, anti-B, anti-A,B, and anti-H Basically, NO forward reaction and POSITIVE reverse Resolution: test with anti-H lectin (Bombay’s don’t have H and will not react)

Finding the problem… Forward type tests for the antigen (red cell) Reverse type tests for the antibody (serum) Identify what the patient types as in both Forward & Reverse Groupings Is there a weaker than usual reaction? Is it a missing, weak, or extra reaction??

Resolving ABO Discrepancies Get the patient’s history: age Recent transplant Recent transfusion Patient medications The list goes on….

Let’s practice !

Example 1 Anti-A Anti-B A1 Cells B Cells 3+ 1+ Problem: Causes: 1+ Problem: Reverse grouping, weakened patient antibody Causes: Age related or weakened immune system Resolution: Incubate at Room Temperature 15-30 minutes and respin. Check patient history. Problem: Causes: Resolution:

Example 2 Anti-A Anti-B A1 Cells B Cells 3+ 1+ 4+ Problem: Causes: 4+ Problem: 1+ reaction with anti-B. Appears to have additional antigens. Causes: Acquired B antigen Resolution: Patient history – bowel obstruction, carcinoma of colon/rectum. (E. coli) Problem: Causes: Resolution:

Example 3 Anti-A Anti-B A1 Cells B Cells 2+ 0+ 1+ 4+ Problem: Causes: Problem: Weak forward with anti-A and 1+ reaction with A1 cells Causes: 1) Subgroup of A (A2 with anti-A1) 2) unexpected cold reacting antibody to antigen on reagent A1 cells Resolution: 1) test patient cells with anti-A1 lectin and with patient serum test with A2 cells 2) an unexpected cold antibody would be detected in the antibody screen Problem: Causes: Resolution:

Example 4 Anti-A Anti-B A1 Cells B Cells 3+ Problem: Causes: 3+ Problem: missing antigen in forward grouping. Patient appears as group A in reverse grouping Causes: A subgroup Resolution: extend incubation time because this may enhance the reaction. Test with a polyclonal or monoclonal blend of anti-A,B (may contain subgroup antigens)….. Problem: Causes: Resolution:

Example 4 Anti-A,B Patient RBC 1+ Probably a subgroup of A (Ax) if the result was negative (0), adsorption or elution studies with anti-A could be performed (these will help determine what A antigens)

Example 5 Anti-A Anti-B A1 Cells B Cells 2+mf 3+ Problem: Causes: 2+mf 3+ Problem: strength of anti-B is weaker than expected; reverse indicates a group B individual Causes: Group B individual transfused with group O cells Resolution: recent transfusion? Bone marrow/stem cell transplant? Find what ABO type the patient was prior to transfusion Problem: Causes: Resolution:

Example 6 Anti-A Anti-B A1 Cells B Cells 4+ 1+ Problem: Causes: 1+ Problem: Forward shows AB individual, Reverse shows weaker “extra” reaction with B cells (looks like a group A) Causes: Possible cold allo- or autoantibody (patient may have an antibody to another blood group system; A1 and B cells may have the antigens to these antibodies) (allo: P, M, N, Lewis) (auto: I or IH) Resolution: screen for antibodies using Screening Cells and an autocontrol (we’ll talk later about Ab screens) Problem: Causes: Resolution:

Example 7 Anti-A Anti-B A1 Cells B Cells Problem: Causes: Resolution: Problem: Reverse grouping, missing patient antibody (probably group O with no antibodies) Causes: Age related or weakened immune system Resolution: Incubate at Room Temperature 15-30 minutes and respin. Check patient history. Problem: Causes: Resolution:

Screening Cells (I and II) Example 6 Screening Cells (I and II) Autocontrol (AC) Conclusion Patient Serum 1 Pos Neg Cold alloantibody Patient Serum 2 Cold autoantibody if alloantibody – antibody ID techniques if autoantibody – special procedures (minicold panel, prewarming techniques); no prior transfusions. If they have had a recent transfusion, then it could be an alloantibody.

References Rudmann, S. V. (2005). Textbook of Blood Banking and Transfusion Medicine (2nd Ed.). Philadelphia, PA: Elsevier Saunders. Blaney, K. D. and Howard, P. R. (2009). Basic & Applied Concepts of Immunohematology. St. Louis, MO: Mosby, Inc.